Efficient insertion of exogenous nucleic acid into the chromosomal and extra-chromosomal nucleic acid of cells is desired in the art of molecular biology to identify chromosomal regions involved in expressing or regulating expression of peptides and proteins. This same technology is also advantageously used in developing new therapeutic and pharmacologic agents.
One common method relies upon in vivo Tn5 mutagenesis to insert polynucleotides of interest into cellular DNA and to construct libraries of cells that contain inserted polynucleotides at random or quasi-random locations. Existing in vivo Tn5 mutagenesis methods require target cells to encode transposase, either natively or from an introduced expression construct. Accordingly, it can be necessary to construct a suitable expression system appropriate to each target cell type. This can be time consuming, and requires extensive knowledge of the requirements of each target cell type.
In many cases, the gene that encodes transposase is encoded by an active transposon, which can continue to transpose in a target cell after the initial desired mutagenesis step. Such undesired residual transposition is undesired in that it complicates the analysis of insertional mutant libraries.
Furthermore, many techniques for in vivo Tn5 mutagenesis rely upon a complex biological mechanism for introducing exogenous DNA into the target cells, such as bacteriophage lambda transducing phage or a conjugating plasmid. It would be desirable to avoid requiring such complex biological systems.
Shoji-Tanaka, A., et al., B.B.R.C. 203:1756-1764 (1994) describe using purified retroviral integrase to mediate gene transfer into murine cells.
Kuspa, A. and W. F. Loomis, P.N.A.S. U.S.A. 89:8803-8807 (1992) and others have described specifically integrating a plasmid linearized with a restriction enzyme into a genomic restriction site by electroporating enzyme-cut nucleic acid along with the cleaving enzyme into target cells.